Maternal biomarkers of endothelial dysfunction and pregnancy outcomes in women with and without HIV in Botswana

Background Women living with HIV-1 (WLHIV) are at higher risk of having an adverse birth outcome, but the underlying mechanism(s) are unknown. We hypothesized that HIV-associated endothelial activation could adversely impact placental function and lead to impaired fetal growth or stillbirth. Methods We used stored samples from WLHIV and HIV-negative women who had enrolled during pregnancy in the observational Botswana Tshipidi cohort. Written informed consent was obtained from the participants. We measured plasma levels of markers of endothelial activation (soluble vascular adhesion molecule 1 [VCAM-1], intercellular adhesion molecule 1 [ICAM-1] and E-selectin) from samples taken during pregnancy. We compared log10 biomarker levels by maternal HIV status and by the timing of ART initiation (ART prior to conception vs. during pregnancy; ART prior to sample collection vs. no ART prior to sampling) using t-tests and the Kruskal-Wallis rank test. We evaluated the association between these biomarkers and adverse birth outcomes (composite of stillbirth or small for gestational age [SGA]) using univariate and multivariate log-binomial regression controlling for maternal age (continuous) and timing of ART start. We also used generalized linear models (GLM) to evaluate the association between continuous birthweight (in grams) and gestational age (in weeks) and markers of endothelial dysfunction, adjusting for maternal age (continuous) and timing of ART relative to sample collection. Results Specimens collected before delivery were available for 414 women (372 WLHIV and 42 HIV-negative women), with a median age of 28 years and median gestational age at sample collection of 30 weeks (range 26, 35 weeks). WLHIV had significantly higher median VCAM1 (p = 0.002) than HIV-negative women, but HIV-negative women had higher median ICAM1 (p = 0.01); e-Selectin levels did not differ by maternal HIV status. Women starting ART during pregnancy had higher log10 VCAM1 levels than those on ART before conception, regardless of whether the sample was collected before (p = 0.02) or after (p = 0.03) ART initiation. However, ICAM1 and e-Selectin did not differ significantly by ART status or ART timing. Ninety-eight women (91 WLHIV and 7 HIV-negative), or 9 (2%) and 89 (22%) included in this study, had a stillborn or SGA baby respectively. Univariate and adjusted analyses did not show significant associations between levels of any of the biomarkers with these adverse birth outcomes. However, lower birthweight (p = 0.03) and lower gestational age at delivery were associated e-Selectin and ICAM (p = 0.008), respectively. Conclusion Maternal HIV infection and lack of ART (or recent ART initiation) were associated with one marker of greater endothelial activation (VCAM-1), but not with other markers (ICAM-1 nor E-selectin) in pregnancy. e-Selectin was associated with lower birthweight and every unit increase in log ICAM-1 at delivery was associated with lower gestation age at delivery.


Methods
We used stored samples from WLHIV and HIV-negative women who had enrolled during pregnancy in the observational Botswana Tshipidi cohort. Written informed consent was obtained from the participants. We measured plasma levels of markers of endothelial activation (soluble vascular adhesion molecule 1 [VCAM-1], intercellular adhesion molecule 1 [ICAM-1] and E-selectin) from samples taken during pregnancy. We compared log 10 biomarker levels by maternal HIV status and by the timing of ART initiation (ART prior to conception vs. during pregnancy; ART prior to sample collection vs. no ART prior to sampling) using t-tests and the Kruskal-Wallis rank test. We evaluated the association between these biomarkers and adverse birth outcomes (composite of stillbirth or small for gestational age [SGA]) using univariate and multivariate log-binomial regression controlling for maternal age (continuous) and timing of ART start. We also used generalized linear models (GLM) to evaluate the association between continuous birthweight (in grams) and gestational age (in weeks) and markers of endothelial dysfunction, adjusting for maternal age (continuous) and timing of ART relative to sample collection.

Results
Specimens collected before delivery were available for 414 women (372 WLHIV and 42 HIV-negative women), with a median age of 28  Introduction Increased access to 3-drug antiretroviral therapy (ART) during pregnancy has dramatically reduced new pediatric HIV infections globally and has improved maternal health [1][2][3][4]. However, women living with HIV (WLHIV) who take ART are at significantly higher risk of having an adverse pregnancy outcome (stillbirth, preterm delivery, and small for gestational age [SGA]) than women without HIV, particularly if ART is started prior to conception [5][6][7][8][9][10][11][12][13][14]. The underlying mechanisms for this increased risk are unknown. Given that more than 1.3 million WLHIV deliver annually [11] it is, therefore, crucial that we study the mechanism(s) underlying adverse birth outcomes in WLHIV, to help inform interventions to improve maternal and child outcomes. Positive HIV status and ART use have both been independently associated with manifestations of endothelial dysfunction, including cardiovascular disease (CVD) [15][16][17][18][19][20][21]. Endothelial dysfunction is a term that covers diminished production/availability of nitric oxide and/or an imbalance in the relative contribution of endothelium-derived relaxing and contracting factors [22]. In the general population, high levels of endothelial adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1), intracellular adhesion molecule 1 (ICAM-1) and selectins (P-selectin or E-selectin) have been consistently associated with endothelial dysfunction [23][24][25][26]. These molecules work in concert to promote the development of atherosclerotic disease, with selectins facilitating activated leukocyte rolling and the adhesion molecules permitting adhesion and passage of leukocytes into the sub-endothelial space [26,27]. During pregnancy, endothelial function is critical for placentation, maternal volume expansion, and placental perfusion. Markers of endothelial dysfunction are associated with adverse birth outcomes in pregnant women without HIV [28], but similar data for pregnant women with HIV do not exist. We hypothesized that vascular endothelial dysfunction due to maternal HIV infection (and/or due to ART taken in pregnancy) adversely impacts placental perfusion during pregnancy, leading to an increased risk of placenta-mediated pregnancy complications such as fetal growth restriction (small for gestational age) and stillbirth. We, therefore, assessed the relationship between (1) HIV, ART and the biomarkers VCAM-1, ICAM-1 and e-selectin, and (2) these biomarkers and adverse birth outcomes.

Study population
Our study utilized stored plasma samples and existing data collected in pregnant women who previously participated in the "Tshipidi" study in Botswana [29]. The Tshipidi study enrolled WLHIV and women without HIV (�18 years of age) during pregnancy or shortly after delivery from 2010 to 2012 and followed mothers and babies for 2 years postpartum to evaluate pediatric neurodevelopmental outcomes. The majority (88%) of women were enrolled before delivery at a median gestational age of 27 weeks. Maternal and infant blood samples were collected at enrollment, delivery, and 1, 6 and 24 months postpartum. Demographic information, HIV history (including the date of diagnosis, antiretroviral [ARV] regimen and date of ARV initiation, and most recent CD4 count and viral load), general medical history, and pregnancy outcome were recorded for all participants. Gestational age was estimated by the last menstrual period (which was ascertained using a combination of maternal self-report and written obstetric records that are completed for all pregnant women during antenatal care and labor/delivery, which nearly universally occur during admission to maternity wards). Written obstetric records also served as the source of birthweight and stillbirth data. Small for gestational age was calculated using estimated gestational age and birthweight.
Women received antiretroviral regimens according to Botswana national guidelines at the time. From 2008 to 2012, pregnant women with baseline CD4 count >250 cells/mm 3 and no WHO Clinical Stage 3 or 4 HIV illness received zidovudine. Women with baseline CD4 <250 cells/mm 3 or with WHO Clinical Stage 3 or 4 received 3-drug ART (generally zidovudine + lamivudine + nevirapine); the CD4 threshold was changed to <350 cells/mm 3 in 2012. Multiple gestations were excluded from this analysis.
The Tshipidi study (from which the de-identified data for this analysis came from) was approved by the Botswana Health Research Development Committee and the Office of Human Research Administration at the Harvard T.H. Chan School of Public Health. Mothers provided written informed consent for study participation. The work summarized in this paper was consistent with original study aims, and was also approved by IRBs as an amendment.

Laboratory testing
One of the secondary objectives of the Tshipidi study was to investigate potential immunemediated mechanisms that might explain adverse birth outcomes in HIV-exposed children. We evaluated this objective using stored maternal plasma samples from all participants who enrolled prior to delivery and who had known infant birth weight, gender, and gestational age. More samples were available for WLHIV than women without HIV at the time we conducted sample testing for this study because most plasma samples for women without HIV had been used by other studies. We measured plasma concentrations of endothelial dysfunction biomarkers ICAM-1 (detection range 0.3-20 ng/mL and sensitivity 0.057) ng/mL, VCAM-1 (detection range 6.3-200 ng/mL and sensitivity 1.26 ng/mL) and E-selectin (0.1-8 ng/mL and sensitivity 0.027 ng/mL) using commercial ELISA kits (R&D Inc, Minneapolis, USA), following the manufacturer's instructions [30]. Testing was done at the ISO 15189 accredited Botswana Harvard HIV reference laboratory.

Statistical analysis
Biomarkers were analyzed as continuous variables. Timing of ART relative to sample collection was defined as 1) initiated pre-conception, 2) initiated post-conception, did not start ART by the time of sampling and, 3) initiated during pregnancy sample collection after ART start.
We compared maternal baseline characteristics using the two-sample t-test for continuous variables and Fisher's exact test for categorical variables. Log biomarker levels were compared by maternal HIV status and by the timing of ART initiation and sample collection using t-tests and the Kruskal-Wallis rank test. We also evaluated the association between these biomarkers and the adverse birth outcomes that are more likely to be related to endothelial dysfunction, namely stillbirth or small for gestational age (defined as <10 th percentile weight-for-gestational age according to Intergrowth-21 standards) [31,32] using univariate and multivariate log-binomial regression controlling for maternal age (continuous) and timing of ART initiation. We also used the generalized linear models (GLM) to evaluate the association between continuous birthweight and gestational age and markers of endothelial dysfunction, adjusting for maternal age (continuous) and timing of ART relative to sample collection.

Results
Among 454 women living with HIV and 458 women without HIV who were enrolled in Tshipidi, this analysis includes 372 women with HIV (82%) and 42 women without HIV (9%) who were enrolled in antepartum and had birth outcomes data and stored plasma collected before delivery available. Among women without HIV: women who had samples available for our analysis had slightly lower median gestation age [(39.5 vs 41 weeks; p = 0.001) but were similar in median age (p = 0.50), educational status (p = 0.90) and the number of stillbirths (p = 0.46) compared with HIV-negative women who did not have samples available. Among 372 WLHIV in our study sample, 346 (93%) had a known time of ARV/ART initiation of whom 39 started ART prior to conception, 181 started during pregnancy prior to enrollment (29 on ART and 152 on ZDV monotherapy) and 126 started after study enrollment/sample collection. Among the 26 women with unknown timing of ART start, 3 were on 3-drug ART, 3 on ZDV, 4 ARV naïve and 16 had no records. Table 1 below shows demographics and clinical characteristics of pregnant WLHIV and pregnant women without HIV. Women living with HIV were older, less educated and had lower income.  Fig 1B. No difference was observed in e-selectin levels by maternal HIV status (Fig 1C).

Markers of endothelial dysfunction by maternal HIV status and timing of ART initiation
Endothelial biomarker level by receipt and timing of ART initiation and sample collection in relation to pregnancy is shown in Fig 2 (initiated pre-conception, initiated post-conception, or did not start ART by the time of sampling). ICAM-1 and e-selectin levels were not significantly different by the timing of ART relative to sample collection (Fig 2B and 2C). However, women starting ART during pregnancy had statistically significantly higher mean log 10 VCAM1 levels (2.92 ng/mL; 95%CI:2.88-2.95) than women who were on ART before conception, in both the samples that were collected before (2.83 ng/mL; 95%CI: 2.77-2.88; p = 0.02) or after (2.91 ng/mL; 95%CI: 2.87-2.944;p = 0.03) ART initiation, Fig 2A.

Markers of endothelial dysfunction and adverse birth outcomes
Univariate and adjusted analyses did not show significant associations between levels of any of these biomarkers and the adverse birth outcomes of interest (stillbirth or SGA). Overall, 9 (2%) pregnancies ended in stillbirth and 89 (22%) with infants born SGA. . Every 1 unit increase in log e-Selectin (ng/mL) was associated with lower birthweight (β: -0.07, p = 0.03) and every 1 unit increase in log ICAM-1 (ng/mL) at delivery was associated with lower gestational age (weeks) at delivery (β: -0.06, p = 0.008), regardless of HIV status.

Discussion
We evaluated whether maternal HIV status or timing of ART initiation was associated with biomarkers of vascular endothelial dysfunction during pregnancy and whether levels of these biomarkers were associated with SGA or stillbirth. We found that positive maternal HIV status was associated with significantly higher VCAM-1 levels, an indication of greater endothelial activation; however, positive maternal HIV status was not associated with increased levels of ICAM-1 or e-selectin. None of these three biomarkers were associated with the occurrence of stillbirth or SGA (although there we did observe a non-significant trend toward a higher risk of these adverse pregnancy outcomes with higher VCAM-1 levels, and we had limited power to evaluate stillbirth as an outcome). Consistent with other studies for non-pregnant adults, median VCAM-1 levels were elevated in pregnant WLHIV compared with pregnant women without HIV [26][27][28], and there was no difference in e-selectin level by HIV status [26]. In the general population, elevated VCAM-1 has been consistently associated with endothelial dysfunction. Unlike prior data from non-pregnant adults [23,26], we found that median ICAM1 levels were higher in women without HIV compared with WLHIV. The reasons for this difference are not clear. ICAM-1 is an important molecule in immune-mediated and inflammatory processes and functions as a co-stimulatory signal which is important for the trans-endothelial migration of leukocytes and the activation of T cells [33,34]. Studies of ICAM-1 in women with HIV show no difference in the levels of ICAM-1 in normal pregnancy compared to non-pregnant women, but significantly increased levels of ICAM-1 in women with pregnancy-induced hypertension, especially preeclampsia [35,36].
We hypothesized that HIV-associated endothelial dysfunction would lead to a poorly functioning placenta and that there would be an increased prevalence of adverse birth outcomes  associated with abnormal placentas, including SGA and stillbirth. However, we found no association between these outcomes and biomarkers of endothelial dysfunction. This could indicate that the biomarkers we chose are not the most specific for endothelial dysfunction in pregnancy, or we may have lacked the power to detect differences given the relatively small study sample size.
Our study had several limitations. Our sample size was small for detecting associations with infrequent outcomes such as stillbirth. We could not measure all biomarkers of endothelial dysfunction and could not determine the causal relationship between endothelial dysfunction and adverse birth outcomes. However, our data suggest a possible role of e-selectin and ICAM-1 in lower birthweight and gestation age at delivery, respectively. This possible role of e-selectin and ICAM-1 in lower birthweight and gestation age at delivery is regardless of HIV status because there were no differences in these parameters by HIV status.
Not all stillbirths or SGA would result from placental dysfunction. Nevertheless, we believe that this study generated valuable pilot data in an area that has not been investigated thoroughly.

Conclusion
Our study did not support endothelial activation as a mechanism for adverse birth outcomes in WLHIV, but further research is needed given the limitations and small sample size in this study.